New Tools for Detection of Leptospira in Horses

Abstract Research Plan In Scientific Terms: Scientific Importance: Leptospira infection is associated with abortion, premature birth, weak foals and recurrent uveitis and is a periodic source of loss to the equine industry in Kentucky and elsewhere. Although vaccination is widely used in other domestic animals, a licensed vaccine is not available for the horse in part because of economic consideration and avoid of sensitization. Instead, control is based on identification of seropositive animals and judicious use of antibiotic. The MAT, fluorescent microscopy, culture and identification currently used for diagnosis are labor intensive and require experienced staff in a specialized laboratory. New approaches are feasible based on our recent discovery of several novel leptospira proteins strongly reactive with sera of aborted mares. The respective genes have been cloned and expressed in E. coli in our laboratory. We hypothesize that simple, sensitive and rapid ELISA and PCR methods for detection of leptospira infection in the horse may be- developed using one of the newly discovered proteins and primers specific for pathogenic leptospires. The specific aims are to " Compare the specificity and sensitivity of LigA, Lk73.5, Qlp42, Lk48 and Lk62 recombinant proteins in ELISA for detection of leptospira specific antibodies in horse serum. " Develop a PCR based method for leptospira detection in horse urine, eye and amniotic fluid. " Develop a PCR based method for differentiation of maintenance (L. bratislava) and accidental leptospira (L. kennewicki, grippotyphosa and other) infections of horses. The laboratory tests developed in this study will improve detection of leptospira infection and thus provide more effective means of studying transmission and implementing control measures. Importance to the Equine Industry: Leptospirosis is a continuing threat to the horse breeder causing loss due to abortion and stillbirth. Chronic infection may lead to recurrent uveitis, a major cause of equine blindness. Control of the disease in the horse is solely based on the prophylactic and therapeutic use of antibiotics. The overall goal of this project is the development of simple and rapid means of detecting infection using ELISA to detect seropositive animals and PCR to detect leptospira in body fluids of seropositive horses. These tools will be valuable in investigation of the source and modes of transmission of L. interrogans serovar Pomona type kennewicki and other leptospira on breeding farms in Kentucky and elsewhere. They will also help in studies of the pathogenesis of and the efficacy of antibiotic prophylaxis. Thus the equine industry will benefit by reduction of loss due to leptospira infection as a result of improved detection and a control of transmission. Abstract of Research Plan in Lay Language: Scientific Importance: Leptospirosis causes loss of foals due to abortion and stillbirth, blindness and cost of veterinary care. Control refers both on antibiotic prophylaxis and management measures based on identification of seropositive animals by MAT and on presence of leptospira in body fluids using FA and culture. This proposal is aimed at developing more simple, reliable and rapid methods of diagnosis. It is based on ELISA for identification of seropositive animals and PCR for detection and identification of leptospira in body fluids. It will exploit our previous discovery of several novel immunoreactive leptospira proteins and associated DNA sequence for use in design of leptospira specific primers for PCR. Importance to the Equine Industry: ELISA for detection of antibodies and PCR for detection of leptospira would be improved substitutes for existing methods. Control of leptospira transmission and veterinary management of infected mares would be greatly facilitated by more sensitive and specific tests.